Cell Imaging DOI: 10.1002/ange.200801965 Three-Coordinate Ligand for Physiological Beryllium Imaging by Fluoresence** Himashinie V. K. Diyabalanage, Kumkum Ganguly, Deborah S. Ehler, Gavin E. Collis, Brian L. Scott, Anu Chaudhary, Anthony K. Burrell, and T. Mark McCleskey* Beryllium is an extremely light, stiff metal with a wide spectrum of applications in industry. Pure beryllium metal, alloys, salts, as well as metal oxides have been extensively utilized in nuclear, aerospace, and electronic industries. [1] However, Be and its compounds are highly toxic, and exposure to particulate Be can lead to chronic beryllium disease (CBD). A recent NIOSH (National Institute for Occupational Safety and Health) report estimates that 26 500 current DOE and DOD employees and up to 106 000 workers in the private sector of the United States have potentially been exposed to beryllium. [2] CBD is a cell-mediated immune response characterized by the development of lung granulo- mas and progressive pulmonary fibrosis, which occurs in up to 6–20% of subjects exposed to beryllium or its salts. [3–6] Even though the nature and effects of CBD have been well-studied, how beryllium triggers such a specific immune response remains a mystery. [5,7] The ability to track Be in cellular studies could provide very useful information on how the immune system is able to selectively recognize the Be 2+ cation and why the immune response continues into granuloma formation. Selective fluorescent indicators have been reported, and an ASTM approved fluorescent method for Be detection has recently been commercialized. [8–12] These advances have made it possible to detect low levels of Be with small sample sizes, providing the ability to determine Be concentrations in Be–protein binding experiments. However, to our knowledge none of the reported fluorescent sensors have been shown to work in a physiological phosphate medium. A selective fluorescent sensor capable of working in a phosphate media would open the possibility for imaging in a biological system. Such a beryllium-staining agent would open up the possibility for a wide range of biological experiments to gain further insights into the understanding of how CBD develops and advance our understanding of the lung immunology in general. Previous research has included binding of beryllium by bidentate ligands to make [BeL] and [BeL 2 ] species with chelating ligands such as chromotropic acid and 2,3-dihy- droxybenzoic acid, which was initially designed for polynuc- lear [Be 2 L] complexes. [13–18] Recent work has suggested that Be binding occurs through the displacement of strong hydro- gen bonds. [19] Strong hydrogen bonds occur when the distance between the two heteroatoms, typically OÀH···O or OÀH···N, is shorter than the sum of the van der Waals radii and the energy barrier to hydrogen transfer between two atoms is on the order of the O À H vibrational zero-point energy. The strong hydrogen bond provides two advantages. First, the O···X distance in a strong hydrogen bond is in the range 2.4– 2.8 , which brings two oxygen atoms into a predefined chelating site for the beryllium atom that corresponds very well to the intraligand oxygen–oxygen atom distances of 2.26 to 2.86  observed in known beryllium structures in the Cambridge Structural Database. [20] Second, the strong hydro- gen bond provides a low barrier pathway to displace the proton without breaking a strong covalent OÀH bond by shifting the proton to the more acidic site as Be interacts with the basic oxygen center. [21] We have used this strategy to select a new tridentate fluorescent agent for binding beryllium. Polypyridines have been widely used as polydentate chelating ligands. Among the polypyridines, the coordination chemistry of polydentate chelating ligands containing mixed pyridine–phenol donors is a very popular target of study. [22–25] The ligand 2,6-bis(2-hydroxyphenyl)pyridine (BHPP) was chosen because it has two basic phenol ligands that could potentially form strong hydrogen bonds with the central pyridine ring. The pyridine ring provides an acidic site that acts as low kinetic barrier pathway to shuttle the protons off the phenol rings, and the two basic phenolates should bind strongly to the Be center. The resulting tridentate [Be(bhpp)] complex was intended to limit the kinetic accessibility of the Be center in phosphate medium. BHPP has recently been studied for its binding to Cu and Zn in nonaqueous media. [26] Herein we demonstrate that this ligand is able to bind Be selectively at neutral pH in the presence of phosphate and is a promising candidate for cellular tracking of Be. BHPP was synthesized by a modified literature procedure utilizing a Suzuki coupling to increase purity of the final product. [27] The proton NMR spectrum of the free BHPP ligand in DMSO shows a sharp singlet at d = 12.31 ppm with an integrated area of 2 for both of the phenol protons. This large downfield shift is indicative of protons that are involved [*] Dr.H. V. K.Diyabalanage,D. S.Ehler,Dr.B. L.Scott,Dr.A. K.Burrell, Dr. T.M. McCleskey Materials Physics and Applications Division Los Alamos National Laboratory, MS J514 Los Alamos NM 87545 (USA) Fax: (+ 1)505-667-9905 E-mail: tmark@lanl.gov Dr. K. Ganguly, Dr. A. Chaudhary Biosciences Division Los Alamos National Laboratory MS M888 Los Alamos NM 87545 (USA) Dr. G. E. Collis CSIRO Molecular and Health Technologies Clayton VIC, 3169 (Australia) [**] This work was supported by the Laboratory Directed Research and Development program (LDRD) at Los Alamos National Laboratory. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.200801965. Zuschriften 7442 # 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2008, 120, 7442 –7444